Commercial glucose isomerase is really a xylose isomerase (D-xylose ketol isomerase, EC 5.3.1.5), an intracellular enzyme that catalyzes the isomerization of D-xylose to D-xylulose. However, the practical significance of the enzyme stems from the fact that the xylose isomerase can use either D-xylose or D-glucose as substrates. The primary industrial use for glucose isomerase is in the synthesis of high fructose corn syrups used as sweeteners. More than 15,000 million pounds of these sweeteners are produced annually, and production levels continue to increase (1-3). Therefore, the xylose isomerase is commonly referred to as glucose isomerase. When we refer to "glucose isomerase," we are referring to the D-xylose ketol isomerase.
All glucose isomerases known and studied to date, including those used in the present industrial processes for making fructose, exhibit considerably lower K.sub.m values for their natural substrate, D-xylose, than they do for D-glucose. (A lower K.sub.m value indicates that less substrate is needed for half-maximal reaction velocity.) In addition, the maximum activity (V.sub.max) of the enzyme for D-xylose is considerably higher than it is for D-glucose. ( A higher V.sub.max value indicates a higher rate of reaction at a saturating substrate concentration.) It would be an advantage, therefore, in the industrial production of fructose to have glucose isomerases with increased affinity for D-glucose as a substrate.
The commercial value of glucose isomerases as a biocatalyst has stimulated research on the structure and function of the enzyme and on the organization and regulation of the glucose isomerase gene in different organisms. (1,4-9) Complete nucleotide sequences and predicted amino acid sequences have been determined for glucose isomerase from Escherichia coli, (10,11) Bacillus subtillis (12,13), Salmonella typhimurium (14), Ampullariella sp.(15), Streptomyces violaceoniger (16), Streptomyces olivochromogenes (7), Streptomyces griseofuscus (17), Arthrobacter (18) and Clostridium thermosulfurogenes (19,20). In addition, three-dimensional structures have been resolved for glucose isomerases from Streptomyces rubiginosus (21), Streptomyces olivochromogenes (22) and Arthrobacter strain B3728 (18,23,24). SEQ ID NO: 1 is predicted amino acid sequence of glucose isomerase from Clostridium thermosulfurogenes.
Comparison of these sequences demonstrates that glucose isomerases from different sources share considerable amino acid sequence homology (17,20,22). SEQ ID NO: 2 is a consensus amino acid sequence we have derived from the sequences disclosed above. An Xaa symbol in SEQ ID NO: 2 indicates a position at which there is no consensus. The consensus sequence is displayed so that the first amino acid of the consensus sequence corresponds to the first amino acid sequence of the Clostridium thermosulfuorgenes sequence in SEQ ID NO: 1. For the Clostridium sequence to fit the consensus sequence, an extra residue must be inserted in the Clostridium sequence after amino acid residues 68 and 419.
Therefore, similarities in the molecular structure and catalytic mechanisms should exist among glucose isomerases from different organisms. In support of this proposal, amino acid residues such as His.sub.101, Thr.sub.141, Val.sub.186, and Trp.sub.139 of the Clostridium enzyme, that were predicted by X-ray diffraction studies of the Arthrobacter enzyme to be part of the active center, and amino acid residues Glu.sub.232 and Glu.sub.268, that were predicted to bind metal ions essential for activity, are highly conserved in the species compared above (19-22).
It was thought initially that enzymatic isomerization of xylose and glucose proceeds via a cis-enediol intermediate and is accomplished by proton transfer with a histidine residue acting as a general base and attracting the proton from the C.sub.1 hydroxyl of the substrate (6,25,26). The analogy of glucose isomerase to triose phosphate isomerase, for which the base-catalyzed enediol mechanism has been demonstrated (25), and some indications from X-ray crystallographic studies (6) seemed to support this hypothesis. The early mutagenesis studies have, therefore, been interpreted as indicating that His.sub.101 was the residue acting as the essential base in the isomerization reaction (10).
However, glucose isomerase exhibits properties very distinct from those of triose phosphate isomerase, which argues that the two enzymes are unlikely to employ the same catalytic mechanisms. Moreover, recent crystallographic studies have indicated that the position of the substrate bound to glucose isomerase is different from the position suggested earlier and, therefore, the essential histidine residue (His.sub.101 in the Clostridium enzyme) is positioned too far from the C.sub.1 and C.sub.2 atoms of the substrate to be able to attract protons from their hydroxyl groups. It was suggested instead that the isomerization proceeds via a metal-catlyzyed hydride shift and the essential histidine is required for either the ring opening or the binding of the substrate (22-24).
The ability to mutagenize a protein at a specific site has enabled scientists to verify some predictions stemming from structural analysis of glucose isomerase. For example, it has been shown that in the E. coli enzyme the His.sub.101 residue is essential for enzymatic activity because when another amino acid is substituted, the resulting protein is inactive (10). The glucose isomerase from Clostridium thermosulfurogenes has proved to be a particularly convenient enzyme for modification by protein engineering because the gene has been expressed in both E. coli and a food-safe host, B. subtilis. The enzyme produced in these hosts could easily be purified in high yields (19). In the Clostridium enzyme, substitutions at the position of several His residues other than His.sub.101 had no effect on enzyme activity (19).
On the other hand, substitution of His.sub.101 by Gln, Glu, Asp or Asn resulted in mutant enzymes that retained 10-14% of the wild type activity, whereas the K.sub.m was not changed significantly. Moreover, the resulting enzymes showed a constant activity at acidic pH below 6.5 (19). This result indicated that the ability of His.sub.101 to be protonated is essential for the activity of glucose and xylose isomerases. However, if this residue had functioned as a simple base its substitution by the non-basic amino acids, such as Gln or Asn would be expected to create an inactive enzyme. Two other possible roles for the His.sub.101 have been suggested: (i) as a catalyst of the ring opening and (ii) as a stabilizer of the transition state (the species of the reaction pathway with the highest free energy).
Determination of the isotope effect of D-[2-.sup.2 H]-glucose on the V.sub.max of C. thermosulfurogenes isomerase has demonstrated that hydrogen transfer, and not ring opening, is the rate-limiting step in the isomerization reaction for both the wild type and the mutant His.sub.101 --&gt; Gln enzyme (20). Furthermore, the x-ray crystallographic studies of the enzyme-substrate complex revealed only the open-chain forms of the substrate bound to the enzyme crystals (21,23,24). These findings strongly suggest that this complex is the most stable species among the reaction intermediates.
Although scientists have studied the active site of glucose isomerase from many different organisms, the art of fructose synthesis still lacks a glucose isomerase with an improved affinity for D-glucose.